Advanced Drug Delivery of Paclitaxel with Thermoresponsive Water-soluble Micelles for Ovarian Cancer

Advanced Drug Delivery of Paclitaxel with Thermoresponsive Water-soluble Micelles for Ovarian Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Advanced Drug Delivery of Paclitaxel with Thermoresponsive Water-soluble Micelles for Ovarian Cancer Ueon sang Shin, Ji-Hye Kang, Ji-Young Hwang, Young-Jae Cho, Sang-Yu Park, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1938906/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract As an anti-cancer drug, paclitaxel (PTX) is known to be effective for treating patients with ovarian cancer. Not only do dose density and side effects of PTX represent a certain difficulty in chemotherapy, but also cause problems related to its poor water solubility during the administration. From the dose-density concept, we hypothesized that continuous release of PTX could increase the duration of exposure to tumor cells and may be an effective ovarian cancer treatment for patients. Herein, we developed highly water-soluble and multi-temperature-responsive PTX delivery systems to treat ovarian cancer. This was accomplished by entrapping the hydrophobic PTX in poly(NIPAAm-co-BVIm) micelles engineered to deliver drugs (pNIB/PTX). Specifically, the pNIB/PTX-3 complex containing 74 μg PTX loaded at 25 °C showed a rapid release of PTX initially (5.9 %/day) during the first five days at 37 °C. The release rates decreased (1.1 %/day) over the following month, indicating a multi-step release. In orthotopic ovarian cancer mouse models, the pNIB/PTX complex resulted in a six-fold greater therapeutic effect with a single intraperitoneal injection than weekly PTX treatment alone in vitro using two human ovarian cancer cell lines, HeyA8 and SKOV3ip. Moreover, the tumor weights in HeyA8 and SKOV3ip1 models were remarkably decreased with the pNIB/PTX complex compared to the controls. The good water solubility and temperature-dependent release of PTX carriers are highly effective for short-term and long-term delivery. This multi-step drug delivery may be used as a potential candidate for the treatment of patients with ovarian cancer. drug delivery system (DDS) paclitaxel (PTX) temperature-responsive polymeric micelle ovarian cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Statement Of Significance Herein, we developed highly water-soluble and multi-temperature-responsive paclitaxel (PTX) delivery systems for the treatment of ovarian cancer. This was accomplished by complexing the hydrophobic PTX with poly(NIPAAm-co-BVIm) polymeric micelles. In vitro and in vivo analysis showed that our PTX carriers resulted in a six-fold greater therapeutic effect than the only PTX in human ovarian cancer cell and the single injected mice showed similar survival to the mice that were injected weekly with PTX alone with significantly decreased tumor weights. Our multi-step drug delivery system could be novel strategy for the treatment of patients with ovarian cancer. Introduction Epithelial ovarian cancer is the most lethal disease among various gynecologic malignancies as it is typically diagnosed in the advanced stages. Almost 70~80 % of patients with ovarian cancer are diagnosed with advanced stage including III or IV. These patients diagnosed with metastatic ovarian cancer (stage III) have only a 39% 5-year-survival rate (statistic in the United States from 1975 to 2017) [1]. Although most patients with advanced-stage ovarian cancer exhibit poor prognosis, more than 70% of patients exhibit a favorable initial response to standard treatment methods, including maximal debulking surgery and adjuvant chemotherapy with paclitaxel (PTX) and carboplatin [2-4]. Taxanes, such as PTX and docetaxel, stabilize polymerized microtubules, resulting in the inhibition of cell division during mitosis [5-9]. PTX and carboplatin administered every three weeks are considered the standard first-line chemotherapy for advanced epithelial ovarian cancer. The concept of dose density is based on the hypothesis that a shorter interval between doses of cytotoxic therapy more effectively reduces tumor burden than dose escalation. The duration of exposure is an important determinant of the cytotoxic activity of PTX. A dose-dense schedule leads to frequent exposure of tumor cells [10, 11]. Dose-dense (i.e., weekly) delivery of PTX may exploit anticancer mechanisms, such as anti-angiogenesis and the induction of apoptosis [12-14]. Therefore, dose-dense chemotherapy with PTX is currently one of the standard adjuvant therapy options for ovarian cancer [15, 16]. From the dose-density concept, we hypothesized that continuous release of PTX could increase the duration of exposure to tumor cells and may be an effective ovarian cancer treatment for patients. In addition, even when administered tri-weekly or weekly, PTX has several significant side effects, including peripheral neuropathy and bone marrow suppression, and represents a major limitation in clinical practice [17, 18]. Not only do dose density and side effects of PTX represent a certain difficulty in chemotherapy, but also cause problems related to its poor water solubility during the administration. Recently, alternative PTX nanoformulations have been developed to minimize or overcome these limitation [19-24]. Continuous release of PTX through a specific drug delivery system (DDS) that includes a water-soluble complex and a multi-step release process via stimuli-responsive characteristics might be able to decrease PTX-related complications in patients. In this study, highly water-soluble and temperature-induced PTX delivery systems were developed and examined for their effectiveness in ovarian cancer treatment in a multi-step drug release process. First, we synthesized a highly water-soluble polymeric micelle of poly(NIPAAm-co-BVIm), named pNIB, and then formed a complex with PTX, which is poorly water-soluble. These complexes, pNIB/PTX, could possess highly water-soluble and temperature-responsive characteristics in treatment of ovarian cancer. Materials And Methods 2.1 Materials N -Isopropylacrylamide (NIPAAm) was purchased from Acros Organics (Geel, Belgium), and purified by recrystallization from hexane (HPLC grade) prior to use. N- vinylimidazole (NVIm), 1-bromobutane, ammonium persulfate (APS), and N , N , N ’, N ’-tetramethylethylenediamine (TEMED) were purchased from Sigma–Aldrich (St. Louis, MI, USA). PTX was purchased from Samyang Co. (Daejeon, Republic of Korea) as a model drug. The Quick Start Bradford Protein assay kit was supplied by Bio-Rad (Hercules, CA, USA). Phosphate-buffered saline (PBS) solution was purchased from Biosesang (Seongnam, Republic of Korea) and used for drug loading and release tests. All other reagents and solvents, purchased from Sigma-Aldrich (St. Louis, MI, USA), were of analytical grade and used without further purification. 2.2 Physicochemical and morphologic characterizations The organic samples were characterized by Fourier-transform infrared spectroscopy (FT-IR) spectroscopy (Spectrum BXII, Perkin-Elmer, Waltham, USA), Zetasizer Nano ZS90 (Malvern, Malvern, UK), and Turbiscan LAB (Formulaction, Toulouse, France). FT-IR spectra were recorded from 4000–500 cm −1 in 16 scans at 4 cm −1 resolution using potassium bromide (KBr) (Fig. S1). The dispersion stabilities of pNIB/PTX samples in phosphate-buffered saline (PBS) were tested by Turbiscan LAB using a pulsed near-infrared light source (880 nm). The sample solutions (0.125 mg/mL) of 50-mm height were scanned lengthwise every 3 h for 9 h at 37. The light transmitted (or backscattered) by the sample solution at 135 °C was measured. The 3D morphologies of pNIB micelles with and without PTX were examined by field-emission scanning electron microscopy (FE-SEM, Hitachi S-4300, Hitachi, Tokyo, Japan) after drying the sample solutions on a coverslip at 25 °C and 37 °C. Samples were sputter-coated with approximately 10 nm of gold at a voltage of 15 kV before analysis. The micelle morphologies of the pNIB/PTX micelles with and without PTX in the dry state were characterized by FE-SEM. Each sample solution (0.1 mg) was placed onto a cover slip and then dried at each temperature. 2.3 Preparation of pNIB micelles and PTX complexes The 1-butyl-3-vinyl imidazolium bromide- (BVIm) and pNIPAAm-based ionic copolymer poly(NIPAAm-co-BVIm) or pNIB were synthesized as described previously (see the 1 H-NMR spectrum of the pNIB copolymer in Fig. S1) [25]. Hydrophilic NIPAAm and BVIm monomers were dissolved in deionized water (DW), APS solution (10% (w/v)) and TEMED were added to the solution as the initiator and activator, respectively. After completion of the reaction, all possible impurities were carefully removed by performing the following steps: i) extraction of organic impurities from the aqueous solution with methylene chloride, ii) removal of inorganic impurities by dialysis (membrane tubing, molecular weight cutoff 12,000–14,000 Da, Spectrum Laboratories, Savannah, GA, USA) against DW, and iii) repetitive precipitation in water through changes in temperature between 4 and 60 °C. After freeze-drying, the purified copolymer product, pNIB, which looks like white cotton wool, was obtained at a yield of ~96.5 wt%. The number- and weight-average molar masses ( Mn and Mw, respectively) of the copolymer appeared to be approximately 1353 and 2001 g/mol, respectively, and showed a molar mass distribution ( Mw / Mn ) of 1.47. Water-soluble pNIB/PTX complexes were prepared by dissolving 0, 100, 125, 150, 175, and 200 μg PTX in 3 mL of ethyl alcohol containing pNIB (1.0 mg) in an ultrasonic bath at 25 °C for several minutes. Powders of the pNIB/PTX complexes, named pNIB/PTX-1, -2, -3, and -4 in this study, were obtained after filtration and vacuum evaporation of ethyl alcohol at room temperature (Table 1). Table 1. Summary data for formation of the pNIB/PTX complex micelles. Sample name pNIB used ( μg) PTX feeded ( μg) PTX loaded ( μg) pNIB 1000 0 0 pNIB/PTX-1 1000 100 53.38 ± 3.30 pNIB/PTX-2 1000 150 64.78 ± 2.05 pNIB/PTX-3 1000 175 74.19 ± 2.77 pNIB/PTX-4 1000 200 80.16 ± 5.30 2.4 Determination of loading and releasing amounts of PTX The maximum amount of PTX that could be loaded within pNIB copolymer micelles at 37°C was determined via solubility (or solution stability) tests in PBS by observation and using TurbiScane LAB. Specifically, 3.0 mg of pNIB/PTX (pNIB/PTX-1, -2, -3, and -4) were separately dissolved in 48 mL PBS solution at room temperature. In vitro PTX loading and release tests were performed using HPLC calibration curves (Fig. S1). Samples were filtered through a polytetrafluoroethylene (PTFE) membrane filter (JGWP01300, Ominipore™, Millipore, Burlington, MA, USA) with 0.20 μm pores and equipped with a syringe. For the release test, 10 mg of pNIB/PTX-3 sample was dispersed in 10 mL PBS and incubated in a 37 °C water bath with shaking. The PBS solution was filtered daily for 40 d. After filtration, the filtrates were evaporated and re-dissolved in 1 mL acetonitrile, and the resulting solutions (20 μL) were injected into HPLC system. The amount of PTX from the carrier was measured by high-performance liquid chromatography (HPLC, Shimadzu LC20A Series, Kyoto, Japan) at a flow rate of 1 mL/min using an acetonitrile/water solution (50/50 (v/v)) as the mobile phase. (Waters Spherisorb ODS2 column, particle size 5 mm, 4.6 mm 150 mm) PTX was detected at a wavelength of 227 nm. The PTX loading capacity (LC) of pNIB/PTX-3 was calculated using the following equation: where M t and M 0 are the cumulative amounts of PTX released at time t and the total amount of PTX entrapped, respectively. The experiment was performed in triplicate. 2.5 Measurement of the size distribution and surface charge changes The mean sizes and zeta (ξ) potential values of pNIB and pNIB/PTX micelles under wet conditions were measured at 25 and 37 °C using Zetasizer Nano ZS90. Sample solutions (3.12 μg/mL) were prepared using a mixture of acetic acid (0.2 M) and sodium acetate (0.1 M). The size distribution curves were obtained using Gaussian curves. These were nearly symmetrical around the vertical line, which passed through the maximum in all cases (Fig. S2). The maximum value is the average size, and the standard deviation is the distance between the two ends at the base. The zeta (ξ) potential values were recorded in a pH range of 4–10 or at pH 7 in a mixture of acetic acid (0.2 M) and sodium acetate (0.1 M) using the Zetasizer Nano ZS90 (Fig. S2). The desired pH was adjusted with HCl or NaOH solution, and the pH values were measured using a pH meter (Orion 3 star, Thermo Scientific, Waltham, MA, USA). All measurements were conducted at least three times for each experimental group, and the average values are represented by the standard deviation. 2.6 Determination of the lower critical solution temperature (LCST) values The temperature-responsive behaviors (LCST values) of pNIB/PTX micelles with or without PTX were determined using a UV-vis spectrophotometer (Evolution 300, Thermo Scientific, Waltham, MA, USA), fitted with a temperature control system. Aqueous solutions (0.1% wt) of the copolymer were prepared in PBS at pH 7 in ~1 mL of the prepared solution was added to a polystyrene UV cell located in the cell holder. The percentage transmittance of light at 500 nm was scanned for each sample during heating at a rate of 1 °C per 10 min between 25 and 45 °C. 2.7 In vitro cell viability assay The in vitro cell viability of PTX-free polymer carrier (pNIB), optimized PTX-loaded pNIB (pNIB/PTX-3), and PTX only was evaluated using a Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan) according to the manufacturer’s instructions. Human epithelial ovarian cancer (EOC) cell lines, HeyA8 and SKOV3ip1, were a gift from Dr. Anil K. Sood (Department of Cancer Biology, University of Texas M.D. Anderson Cancer Center, TX, USA), and were maintained in complete media (RPMI 1640) supplemented with 10% fetal bovine serum (FBS) and 0.1% gentamicin sulfate (Gemini Bioproducts, Calabasas, CA, USA) in a 5% CO 2 atmosphere at 37 °C. Prior to the cell viability assay, cell suspensions were seeded at a density of approximately 5 × 10 3 cells per well in 96-well microplates and pre-incubated for 12 h. The UV-sterilized sample solution was prepared by three-fold serial dilution with complete culture medium. Each well was replaced with each respective concentration set (n = 4–8) to a final concentration of 1,500 mg/mL to approximately 0.009 mg/mL. PTX alone was diluted from 450 to approximately 0.003 μg/mL. After the plates were incubated at 37 °C for an additional 48 h, particles and floating dead cells were discarded. The plates were then incubated with 100 μL of 10-fold diluted CCK-8 solution for 4 h. The optical density (OD) was measured at 450 nm using a microplate reader (Molecular Devices, USA). Cell viability rates were calculated using the following equation: where OD control is obtained in the absence of nanoparticles or drugs. All experiments were repeated four to eight times, and the data are presented as mean ± standard deviation (SD). 2.8 Development of orthotopic mouse models for ovarian cancer In vivo experiments were performed to confirm the effects of pNIB/PTX-3 on tumor growth in an orthotopic mouse model using cell lines. Female BALB/c nude mice were purchased from Orient Bio (Seongnam, Republic of Korea). The study was performed in accordance with relevant guidelines and regulations and was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Samsung Biomedical Research Institute (SBRI, Seoul, Republic of Korea). SBRI is an Association for Assessment and Accreditation of Laboratory Animal Care International (protocol no. H-A9-003)-accredited facility and abides by the Institute of Laboratory Animal Resources (ILAR) guidelines. To generate tumors, HeyA8 (2.5 × 10 5 cells) or SKOV3ip1 (1.0 × 10 6 cells) in 0.2 mL Hank's balanced salt solution (HBSS, Gibco, Waltham, MA, USA) were injected into the peritoneal cavity of 8- to 12-week-old BALB/c nude mice. Seven days after the cell injection, 1X pNIB, 4X pNIB, 1X pNIBm/PTX-3 (PTX 120 μg/mouse), 4 X pNIB/PTX-3 (PTX 480 μg/mouse), or PBS only (as a control) was injected into the peritoneal cavity. Mice were monitored daily for tumor development and postoperative complications. Mice were sacrificed on days 35–40 or when the mice seemed moribund. The body and tumor weights of each mouse were recorded. To assess survival, the animals were observed weekly until death. Tumors were fixed in formalin and embedded in paraffin or snap frozen with liquid nitrogen in OCT compound (Sakura Finetek Japan, Tokyo, Japan). Apoptotic cell death was assessed according to the manufacturer’s instructions using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay kit (Promega, Fitchburg, WI, USA). To quantify cell death, the number of TUNEL positive cells was counted in five random fields at 100x magnification, and the percentage of positive cells was calculated [26]. 2.9 Statistical analysis Statistical analyses were generally performed using SPSS ver. 21 (SPSS Inc., Chicago, IL, USA). For in vitro and in vivo analysis, the graphs, calculations, fitting curves and statistical analyses were performed using GraphPad Prism software version 9.0 (GraphPad Software Inc., San Diego, CA, USA). Survival data was plotted via Kaplan-Meier curves and analyzed with the log-rank test. The statistical significance of differences was determined using one-way analysis of variance (ANOVA) for more than two pairs. The level of significance was represented at a P value of < 0.05 in ANOVA. All experiments were repeated at least three times and data are presented as means ± standard deviation (S.D.). Results And Discussion 3.1 Concept of our study Highly water-soluble and temperature-responsive multi-step drug delivery system for the treatment of ovarian cancer was accomplished by entrapping the hydrophobic PTX molecules within the pNIB polymeric micelles through self-assembly (Fig. 1A). After injection of this pNIB/PTX complex into the ovarian cancer-generated mouse model (Fig. 1B), the complex contracts through drug-regulated lower critical solution temperature (LCST), which allows for the following processes: i) a short-term (a few days) temperature-induced fast release and fast diffusion through the large nanopores of swelled micelles (Fig. 1C, step 2) ii) and then a slow continuous release by diffusion over the long term (about a month) (Fig. 1C, step 3). It is also characterized by a multi-step drug delivery system, which allows the LCST to be downgraded as the drug is released, resulting in phased drug release according to contraction (Fig. 1C, step 4). The physicochemical and biological characteristics of the pNIB/PTX complex, including its high solubility in water, temperature-responsive multi-step delivery system, and advanced therapeutic effects in vitro and in vivo could make it favorable for the stepwise delivery of highly hydrophobic drugs. 3 .2 Formation and water-solubility of the pNIB/PTX complexes The anticancer drug, PTX, which is known to be especially effective for ovarian cancer treatment, has limited delivery capacity through hydrophilic body fluids as it is hydrophobic in nature and exhibits extremely restricted solubility in aqueous media [27, 28]. To maximize the delivery efficiency of PTX through aqueous body fluids, water-soluble enhancements and controlled release are necessary [29-35]. To this end, highly water-soluble and temperature-responsive drug-releasable pNIB/PTX -1, -2, -3, and -4 complexes were prepared for use in this study as a PTX delivery system for ovarian cancer therapy (summarized in Table 1). This was accomplished by entrapping hydrophobic PTX simultaneously when ionic and highly water-soluble copolymer micelles of pNIB were formed by self-assembly (Fig. 2A). When the amount of PTX loading on each sample was measured, it was confirmed that the amount of PTX loaded during the formation of pNIB polymeric micelles increased proportionally with the amount of feed. The maximum loading amount of PTX was 80.75 µg per 1 mg pNIB polymer. However, the efficiency compared to the feeding amount can be observed to decrease from 53–40% as the feeding amount increases (Fig. 2B). The Zeta (ξ) potential values of pNIB at pH 4–10 appeared to be in the range +4.8 to +1.8 mV and about +4.2 mV at pH 7, indicating that the cationic character of the pNIB copolymer was maintained in various conditions and that it had high solubility in aqueous solution over the full pH range (Fig. S2). Nevertheless, the surface charge of the pNIB/PTX complex changed in accordance with PTX loading, as shown in Fig. 2C. These results clearly indicate that the presence of water-insoluble PTX in all pNIB/PTX complexes decreased the ionic characteristics. Although much lower than the control pNIB due to the hydrophobic characteristic of PTX, this comparison showed similar zeta (ξ) potential values of approximately 2.7 mV within a margin of error of ± 0.1 mV, indicating that the pNIB/PTX micelle complex was water-soluble after PTX loading. Furthermore, we examinated the dispersion and solubility by recording transmittance at 25 °C and 37 °C for 24 h to determine the stability of each pNIB/PTX solution (Fig. 2D and Fig. S3). During the entire scanning time, no noticeable changes in the light backscattered by pNIB/PTX-1 ~ -3 solutions over time were detected at either 25 or 37 ° C. As the amount of PTX loaded at the two constant temperatures increased, the transmitted light decreased slightly at 25 ° C from ~85% in pNIB/PTX-1 to 70% in pNIB/PTX-4, while at 37 ° C, it decreased significantly from approximately 77% to 45%. This can be explained by the increased hydrophobicity of the solute and the temperature of the solution, which could result in agglomerate formation of colloid (micelle) particles and an increase in the amount of light backscattered. Despite agglomerate formation in the solutions, no precipitate formation was observed in pNIB/PTX-1 ~ -4 in the photographs (Fig. 2D and Fig. S3). These results demonstrate the highly stable and uniform dispersion of pNIB/PTX-1, -2, -3, and -4 complex micelles (or micelle agglomerates) in aqueous solution. Additionally, all samples showed that transmittance increased over time at 37 ° C, indicating that the drug was released and the solubility of pNIB/PTX was improved (Fig. 2D and Fig. S3). Overall, these results should be noted that all samples represent high solubility and dispersion stability, especially for pNIB/PTX-2 and pNIB/PTX-3, which have markedly stable results over time. 3 .3 Temperature-responsive characteristics of the pNIB/PTX complexes To confirm the temperature-responsive characteristics at 25 ° C and 45 ° C and at pH 7.0, the LCST values of pNIB/PTX complex were determined and compared with the LCST value of pNIB (Fig. 3A). This can be explained by the increased turbidity of the pNIB solution due to polymer micelle shrinkage caused by the polymer phase transition in the aqueous solution. In contrast, when the contraction degree of pNIB/PTX micelle complex samples was compared by temperature, it was clearly confirmed that the phase transition temperature (LCST) increased above body temperature as the amount of loaded PTX in the micelle increased (Fig. 3A and 3B). Hydrophobic PTX molecules would be primarily attached to the hydrophobic parts of the pNIB micelles such as a (CH 2 -CH 2 )-backbone and isopropyl groups of the pNIB molecules. This interaction between PTX and pNIB caused steric hindrance inside the micelles, resulting in an increased LCST (Fig. S4). Accordingly, it was confirmed that a linear curve and an R-square value observed at the phase transition temperature according to the amount of loaded PTX were determined by the standard curve in Fig. 3B. Theoretically, the LCST values increased linearly at a rate of 1.16 o C per 10 µg of PTX out of 1 mg pNIB against the amount of loaded PTX. This means that the temperature-responsive characteristics could be easily changed by adjusting the loading concentration. Moreover, the transmittance of the PTX-free pNIB micelles suddenly decreased by approximately 48% at 37 ° C in the LCST range. On the other hand, the transmittance decrease at 37 ° C was only approximately 23% for pNIB/PTX-3. This phenomenon also strongly implies a two-step paclitaxel delivery of pNIB/PTX-3: a temperature-induced fast delivery of PTX (approximately 23% of the loaded drug) at 37 ° C, followed by slow delivery of the remaining 65% PTX through diffusion at the same temperature (Fig. 1 and Fig. 3A). These data are also consistent with the results shown in Fig. 3D. As an effective way to confirm whether pNIB micelles contain PTX, the average micelle sizes of pNIB/PTX-1, -2, -3, and -4 complexes in aqueous solution were measured using a Zetasizer at 25 ° C and 37 ° C and compared to the values of PTX-free pNIB complex with a temperature increase of 25 ° C to 37 ° C, the series of pNIB/PTX samples showed temperature-induced decreases in the average size of the complex micelles (129, 48, 27.9, 4.2 nm for pNIB/PTX-1, -2, -3, -4, respectively). The PTX-free pNIB micelles exhibited the largest drop of approximately 391 nm. These results indicate that the sharp drop in the diameter of the pNIB/PTX series at 25 ° C is caused by the effective integration of hydrophilic pNIB micelles with hydrophobic PTX molecules. When the PTX concentration increased, the hydrophilicity and equilibrium swelling degree of the pNIB/PTX complexes decreased, resulting in a decrease in the diameter. In particular, the temperature-induced decrease in diameter of approximately 27.9 nm observed from pNIB/PTX-3 micelles might be appropriate for multi-step paclitaxel delivery. This result demonstrates fast delivery at 37 ° C, followed by slow delivery by diffusion at the same temperature. In addition, the opposite is possible if the LCST increases as the drug is loaded. As shown in Fig. 3D, the smaller the amount of drugs loaded, the smaller the size of the drug carrier at the same temperature, 37 ° C. This means that as the drug is released, the amount of drug loaded in the micelle decreases, thereby reducing the LCST gradually through a cascade process, further accelerating drug release (Fig. S4). 3 .4 In vitro release profile of PTX from the pNIB/PTX-3 In this respect, pNIB/PTX-3 was selected as a representative model because it has the maximum loading condition considering the loading capacity and efficiency and highly stable uniform dispersion. Most importantly, its LCST is suitable for application in ovarian cancer via multi-step anti-cancer drug delivery. The stepwise temperature-responsive release process of the pNIB/PTX-3 complex was observed by FE-SEM for morphological changes depending on the temperature, with or without PTX (Fig. 4A). Upon increasing the amount of loaded PTX within the pNIB/PTX complex at a constant temperature, the following morphological changes were observed: i) the shape changed spherical particles (pNIB) into a raspberry shape (pNIB/PTX-3) according to the drug loaded, ii) the average size of pNIB/PTX-3 became smaller than that of PTX-free pNIB micelles. In addition, the histogram showing the size distribution of each sample by temperature indicated the temperature range in which the complexes contracted (Fig. 4B). For instance, pNIB contracted to 43.85 nm at 37 o C, which had an average size similar to one at 40 o C (41.65 nm). In contrast, pNIB/PTX-3 contractions of the hydrogel sequentially occurred (87.76 nm, 63.24, and 56.13 nm) at 25 o C, 37 o C and 40 o C. As PTX was loaded, the LCST increased, resulting in a sequential size reduction at increasing temperatures (Fig. 4A, Fig. S5 and Table S1.). This analysis to be a consistent result with the above figure, and it is worth noting that pNIB/PTX-3 has a consistent contraction ratio with Fig. 3A, especially given that it has a contraction ratio of 27.94% from 25 o C to 37 o C. As shown in Fig. 4C and 4D, the temperature-dependent cumulative release amount of PTX was investigated at 37 °C and 40 °C. All samples showed sustained release profiles for up to 40 days. The initial rapid release (0.59 %/day, 4.41 ± 0.04 μg/day from 1 mg pNIB/PTX-3) for the first five days may have been caused by fast diffusion of hydrophobic PTX molecules through the relatively large nanopores of the swelled micelles and relatively weak compressive desorption of PTX molecules through temperature-responsive micelle contraction at 37°C, whereas the relatively slow release (0.11 %/day, 0.81 ± 0.06 μg/day from 1 mg pNIB/PTX-3) for the remaining days may have been caused by the decreased diffusion speed of drug molecules because of reduced loading amount and narrowed nanopores of the micelles, and by relatively fast compressive desorption of PTX because of lowed LCST values of the micelles at the same temperature. At 40 °C, the drug release was markedly more rapid for 5 d (0.63 %/day, 4.68 μg/day from 1 mg pNIB/PTX-3). And also, the cumulative maximum release amounts were more increased (750.1 μg) than 37 ℃ (512.2 μg) for the entire period. Taken together, when the temperature increased near the body temperature (37 °C), the resulting temperature-induced delivery occurred sequentially: fast delivery by size contraction and then slow delivery by diffusion. This process is a multi-step drug delivery process that is required for sustained release. In addition, when the temperature was raised above the LCST (40 °C), the pNIB/PTX complex shrank more, resulting in an instant greater release of the drug. Therefore, the micelle complex can control the amount of drug release, as desired in the right place. 3 .5 In vitro dose-dependent therapeutic effects of the pNIB/PTX-3 To investigate the potential therapeutic effects of pNIB/PTX-3 in vitro , two human ovarian cancer cell lines, HeyA8 and SKOV3ip1, were treated with different concentrations of PTX, pNIB, and pNIB/PTX-3 at 37 ° C for 2 d. Cell viability was assessed using CCK-8 assay (Fig. 5). Exposure to only the PTX-carrier (pNIB) had IC 50 values of 11.89 μg/mL with approximately 75% cell viability in HeyA8 cells and 33.09 μg/mL with about 65% cell viability in SKOV3ip1 cells. The IC 50 values depending on treatment with pNIB/PTX-3 and PTX alone were 1.516 and 233 μg/mL, respectively, with about 70% and 40% cell viability in HeyA8 cells, in which the calculated PTX concentrations were 0.311 μM and 273 μM, respectively (Figure 5A). In SKOV3ip1 cells, the IC 50 values of pNIB/PTX-3 and PTX alone were 3.421 mg/mL (~40%) and 136.2 mg/mL (~30%), respectively, and the calculated PTX concentrations were 0.701 μM and 159 μM, respectively (Fig. 5B). There was a greater reduction in cell viability in SKOV3ip1 cells (~36%) relative to HeyA8 (~55%) with pNIB/PTX-3. HeyA8 cells had an approximate 55% cell viability with 5.556 μg/mL of pNIB/PTX-3 and in the PTX treatment when PTX was loaded at ~1.1 μM and a concentration of PTX was 6.5 μM. This demonstrates that pNIB/PTX-3 in both cell types had six times more therapeutic effect than PTX treatment alone. We observed dose-dependent inhibitory effects of pNIB/PTX-3 on ovarian cancer cell viability. These results suggest that the pNIB/PTX-3 had good therapeutic effects on ovarian cancer cells and could be an ideal candidate material for PTX delivery in human ovarian cancer patients. 3 .6 In vivo assay of the pNIB/PTX-3 for ovarian cancer therapy To assess the potential clinical relevance of the in vitro results, we performed in vivo experiments using ovarian cancer orthotopic mouse models. HeyA8 and SKOV3ip1 ovarian cancer cells were implanted into the peritoneal cavity of female nude mice and 7 days after cell injection, and therapy was initiated according to the following five treatment regimens: control (PBS saline), 1X pNIB, 4X pNIB, 1X pNIB/PTX-3, and 4X pNIB/PTX-3 (Fig. 6A). In the carrier alone group, which included 1X pNIB and 4X pNIB, tumor weight did not differ from the control. Thus, carrier pNIBIm had no influence on the mouse tumor weight. However, mice treated with PTX loaded with pNIBIm, which included 1X pNIB/PTX-3 and 4X pNIB/PTX-3, had significantly decreased tumor weight compared to the control and carrier alone groups using either of the cell types (Fig. 6B and 6C). We also found that there was no significant difference between 1X pNIB/PTX-3 and 4X pNIB/PTX-3. Thus, 1X pNIB/PTX-3 is a sufficient dose for the treatment of tumor-bearing mice. Daily monitoring of animals throughout the course of therapy showed acceptable tolerability with no adverse side effects, such as changes in body weight, mobility, posture, or feeding habits. Because PTX has anti-proliferative and pro-apoptotic activity in cancer cells, we examined tumor cell proliferation and apoptosis using Ki67 expression and a TUNEL assay, respectively, in harvested tumor tissues. Positive staining of Ki67 was significantly decreased in the pNIB/PTX-3-treated group compared to that in the controls (Fig. 7A and 7C, P < 0.01). A TUNEL assay also showed significantly increased apoptosis in the treated groups (Fig. 7B and 7D, P < 0.01). Finally, we evaluated the survival of mice after injection in the SKOV3ip1 mouse model. At 7 d post-injection, therapy was started according to the following six treatment regimens: control, 1X pNIB, 4X pNIB, 1X pNIB/PTX-3, 4X pNIB/PTX-3, and PTX alone. We administered a single i.p. injection to the four pNIB-treated groups, but PTX 120 mg/mouse, i.p. once a week in the PTX alone group. Kaplan-Meier analysis showed that the 1X pNIB/PTX-3, 4X pNIB/PTX-3, and PTX alone groups had significantly increased survival rates compared to the control group (Fig. 8). However, among the three PTX-containing treatment groups, 1X pNIB/PTX-3 and 4X pNIB/PTX-3 showed no significant differences from the PTX alone group. The pNIB/PTX complex showed six times higher therapeutic effect with a single i.p. injection than weekly PTX treatment alone in in vitro experiments using two human ovarian cancer cell lines, HeyA8 and SKOV3ip. Moreover, we found that the singly injected complex significantly decreased the tumor growth in orthotopic mouse models using either of the cell lines. Survival was significantly prolonged in the mice compared to the control and showed survival rates similar to those obtained by weekly injections of PTX alone. These results suggest that a single injection of pNIB/PTX-3 has a survival benefit similar to that of weekly PTX injections. Conclusion Herein, we successfully developed highly water-soluble and multi-step drug-releasing pNIB/PTX complexes by entrapping water-insoluble PTX (53.38–80.16 μg) within highly water-soluble and temperature-responsive pNIB micelles (1.0 mg). While the high colloidal stability and uniform dispersion of pNIB/PTX-1, -2, and -3 complexes in aqueous solution were confirmed, the pNIB/PTX-3 sample showed optimal solution stability and maximum PTX-loading capacity at 37 °C. Highlight temperature-induced multi-step PTX delivery confirmed that the LCST increased above body temperature in accordance with the increasing amount of loaded PTX in micelles. In particular, pNIB/PTX-3 micelle complex are efficient for the temperature-induced fast delivery of the loaded PTX (approximately 23%) at 37 °C, followed by the slow delivery of the remaining PTX (approximately 65%) through diffusion at 37 °C. Furthermore, the average micelle sizes of complex samples, with and without PTX, in aqueous solution from 25–37 °C exhibited a strong reduction in diameter. These results strongly indicate that pNIB/PTX-3 may be controlled by a cascade process involving the gradual reduction of LCST. The SEM images of pNIB/PTX-3 at 25 °C, 37 °C, and 40 °C also appeared very different in terms of particle shape (rough raspberry-shaped and dense particle-shaped, respectively) and diameter (Contraction 30% at 37 °C and 40% at 40 °C). In addition, unlike pNIB, which already exhibited a contraction at 37 °C, progressive contraction was characterized by morphological transition at 25 °C, 37 °C, and 40 °C. During the 40 d in vitro PTX release assay at 37 °C, an initial rapid release (0.59 %/day, 4.41 ± 0.04 μg/day from 1 mg pNIB/PTX-3) was observed for the first five days, which was followed by a slow release (1.1 %/day, 0.81 ± 0.06 μg/day from 1 mg pNIB/PTX-3) for the remaining days. Therefore, the drug was released more rapidly (75.01 μg per 1 mg pNIB/PTX-3) at temperatures higher than the LCST (40 °C) when compared to the drug release at 37 °C (51.22 μg per 1 mg pNIB/PTX-3), resulting in a sudden drug release. Taken together, the highly water-soluble pNIB/PTX-3 complexes demonstrate multifunctional properties, including a high loading amount of PTX, outstanding water solubility, and multi-step drug release in accordance with temperature, and may be used as promising ovarian cancer-target materials. During the in vitro experiments of the pNIB/PTX-3 complex on the two ovarian cancer cell lines, HeyA8 and SKOV3ip1, the PTX carrier complex, pNIB/PTX-3, showed a six times stronger therapeutic effect than PTX treatment alone. These inhibitory effects of pNIB/PTX-3 suggest that the PTX carrier had good therapeutic effects on ovarian cancer cells and could be an ideal candidate biomaterial for PTX delivery. In the orthotopic ovarian cancer mouse models, this complex significantly decreased tumor weight compared to controls in both the HeyA8 and SKOV3ip1 models. Moreover, this singly injected complex significantly prolonged mouse survival compared to controls and showed similar survival to mice treated with weekly injections of PTX alone. These results show that the good water-solubility and temperature-induced multi-step PTX release of the pNIB/PTX complex are highly useful for short-term and long-term delivery of highly hydrophobic anticancer drugs, including PTX, to ovarian cancer cells. Despite the great advantages associated with the use of PTX in cancer therapy, its administration has certain limitations due to its poor solubility and low permeability. In this study, we successfully developed advanced delivery systems based on stimuli-responsive PTX release to address this limitation. Our multi-step drug delivery system showed ovarian cancer-targeting effect, making it a good therapeutic candidate for the treatment of patients with ovarian cancer. Declarations Ethical approval and consent to participate : All animal studies performed in this study were in accordance with relevant guidelines and regulations and was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Samsung Biomedical Research Institute (SBRI, Seoul, Republic of Korea). Consent : not applicable. Consent for publication : Not applicable Availability of data and materials : The datasets used and/or analyzed during the current study are available from the corresponding author, upon reasonable request. Competing interest : The authors declare that they have no competing interests. Funding : This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) [2020R1F1A1072210]. This work was also supported by the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, Republic of Korea, the Ministry of Food and Drug Safety) [KMDF_PR_20200901_0153-2021] and the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) [2020R1C1C1007482]. Author’s contributions : Prof. Ueon Sang Shin, Prof. Jeong-Won Lee : conceived and supervised. Ji-Hye Kang, Ji-Young Hwang, Young Jae Cho : wrote and performed most of the experimental studies. Sang-Yu Park, Jung-Joo Choi, E-Sun Paik : research co-ordination. Acknowledgements : This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) [2020R1F1A1072210]. This work was also supported by the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, Republic of Korea, the Ministry of Food and Drug Safety) [KMDF_PR_20200901_0153-2021] and the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) [2020R1C1C1007482]. References Howlader N NA, Krapcho M, Miller D, Brest A, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA SEER Cancer Statistics Review, 1975-2017. J Natl Cancer Inst Bethesda, MD. 2020. Armstrong DK, Bundy B, Wenzel L, Huang HQ, Baergen R, Lele S, et al. Intraperitoneal cisplatin and paclitaxel in ovarian cancer. N Engl J Med. 2006,354:34-43. Cortez AJ, Tudrej P, Kujawa KA, Lisowska KM. Advances in ovarian cancer therapy. Cancer Chemother Pharmacol. 2018,81:17-38. Raja F, Chopra N, Ledermann J. 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Characteristics, properties and analytical methods of paclitaxel: a review. Crit Rev Anal Chem. 2018,48:110-8. Chakroun RW, Wang F, Lin R, Wang Y, Su H, Pompa D, et al. Fine-tuning the linear release rate of paclitaxel-bearing supramolecular filament hydrogels through molecular engineering. ACS nano. 2019,13:7780-90. Dalela M, Shrivastav T, Kharbanda S, Singh H. pH-sensitive biocompatible nanoparticles of paclitaxel-conjugated poly (styrene-co-maleic acid) for anticancer drug delivery in solid tumors of syngeneic mice. ACS Appl Mater Interfaces. 2015,7:26530-48. Dong C, Zhou Q, Xiang J, Liu F, Zhou Z, Shen Y. Self-assembly of oxidation-responsive polyethylene glycol-paclitaxel prodrug for cancer chemotherapy. J Control Release. 2020,321:529-39. Wu J, Wang Q, Dong X, Xu M, Yang J, Yi X, et al. Biocompatible AIEgen/p-glycoprotein siRNA@ reduction-sensitive paclitaxel polymeric prodrug nanoparticles for overcoming chemotherapy resistance in ovarian cancer. Theranostics. 2021,11:3710. Yan C, Liang N, Li Q, Yan P, Sun S. Biotin and arginine modified hydroxypropyl-β-cyclodextrin nanoparticles as novel drug delivery systems for paclitaxel. Carbohydr. 2019,216:129-39. Zhai J, Luwor RB, Ahmed N, Escalona R, Tan FH, Fong C, et al. Paclitaxel-loaded self-assembled lipid nanoparticles as targeted drug delivery systems for the treatment of aggressive ovarian cancer. ACS Appl Mater Interfaces. 2018,10:25174-85. Supplementary Files Graphicalabstract.png Graphical abstract SupplementaryMaterialusshin12220808.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1938906","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":133898336,"identity":"8a1dfd3b-e4bf-4e1a-bd1a-e45dc893984c","order_by":0,"name":"Ueon sang Shin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIie3RIQvCQBTA8XcIswxX3xU/w8ngNOzDbAizLMuSmmYRrPNbTAQxGCYLlgPr4q5cMgs2dxqFU5vh/uE4Hvy4Bwdgs/1nDoSAfa89yQKA6ZG+fCIjny5+IgBpVJTfEnY5q0YekOzOYyXzNPChWzVkczSQOhmySGCHCzUcFCLm4MaMFMpEXAejDB1eh5zKrAoAEiBNaV5ME9fPJ7cX8a4fSBlyTZBhwuk2qzhg+0phILROnoShuE5pLmLfQcVOuYH02sXoPZvN18vJnq7SYLD2xlKuDOQ9/U0/AZvNZrO99wAoOVPKA6tfvQAAAABJRU5ErkJggg==","orcid":"","institution":"Dankook University - Cheonan Campus","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ueon","middleName":"sang","lastName":"Shin","suffix":""},{"id":133898337,"identity":"74856866-c44e-420c-a2bd-168f3f6bde02","order_by":1,"name":"Ji-Hye Kang","email":"","orcid":"","institution":"Dankook University - Cheonan Campus","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ji-Hye","middleName":"","lastName":"Kang","suffix":""},{"id":133898338,"identity":"624cf5a9-cf60-4c5f-bf3b-23623e217944","order_by":2,"name":"Ji-Young Hwang","email":"","orcid":"","institution":"Korea Institute of Carbon Convergence Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ji-Young","middleName":"","lastName":"Hwang","suffix":""},{"id":133898339,"identity":"b36c685d-3b11-4876-815b-69d443e8f46c","order_by":3,"name":"Young-Jae Cho","email":"","orcid":"","institution":"Samsung Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Young-Jae","middleName":"","lastName":"Cho","suffix":""},{"id":133898340,"identity":"162013e3-396c-4074-9808-e6950807a4e2","order_by":4,"name":"Sang-Yu Park","email":"","orcid":"","institution":"Dankook University - Cheonan Campus","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sang-Yu","middleName":"","lastName":"Park","suffix":""},{"id":133898341,"identity":"4a8410dd-0285-446e-a638-bd1625afa871","order_by":5,"name":"Jung-Joo Choi","email":"","orcid":"","institution":"Samsung Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jung-Joo","middleName":"","lastName":"Choi","suffix":""},{"id":133898342,"identity":"1e07bbb0-7d4f-45e3-b7be-89cb4dc72b07","order_by":6,"name":"E-Sun Paik","email":"","orcid":"","institution":"Kangbuk Samsung Hospital: Kangbuk Samsung Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"E-Sun","middleName":"","lastName":"Paik","suffix":""},{"id":133898343,"identity":"0cff34a4-d51e-4b64-8bb2-1242c9d0d801","order_by":7,"name":"Jeong-Won Lee","email":"","orcid":"","institution":"Samsung Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jeong-Won","middleName":"","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2022-08-07 21:02:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1938906/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1938906/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":26190392,"identity":"a0766801-781e-4f0c-a624-46b7e8869909","added_by":"auto","created_at":"2022-09-07 17:09:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":728166,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of the advanced drug delivery system of pNIB/PTX complex for the treatment of ovarian cancer.\u003c/strong\u003e (A) Highly water-soluble and temperature-responsive multi-step drug delivery system for the treatment of ovarian cancer was accomplished by entrapping the hydrophobic PTX molecules within the pNIB polymeric micelles. (B) The orthotopic ovarian cancer mouse models were generated by injection of human epithelial ovarian cancer (EOC) cell lines into the peritoneal cavity of nude mice. (C) With a single intraperitoneal (i.p.) injection of pNIB/PTX complex, advanced multi-step drug delivery system was triggered as in the followings, the rapid PTX release occurred for a few days (ii) through the initial partial contraction in the abdominal cavity (i), followed by slow drug release over diffusion in the next few weeks (iii). As drug release progressed, remaining amount of initially loaded drug lowered the lower critical solution temperature (LCST), which led to more shrinkage and further drug release for the next couple of months (iv). These PTX carriers resulted in a six-fold greater therapeutic effect than the only PTX in human ovarian cell and the single injected mice showed similar survival to the mice that were injected weekly with PTX alone with significantly decreased tumor weights. Our multi-step drug delivery system showed remarkable ovarian cancer-targeting effect, making it a good therapeutic candidate for the treatment of patients with ovarian cancer.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-1938906/v1/b7f900a567caca28045ec600.png"},{"id":26190677,"identity":"c10ef770-09cd-49e8-a1d5-70a53ac7a8c1","added_by":"auto","created_at":"2022-09-07 17:14:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":276673,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of the pNIB/PTX micelle complexes.\u003c/strong\u003e (A) Chemical structures of the pNIB polymeric carrier and a schematic illustration of the preparation of the pNIB/PTX complexes, which are formed when the drug is trapped through self-assembly of polymeric micelles. (B) The loading capacity of the PTX, which was entrapped in pNIB micelles, was linearly increased, while loading efficiency was saturated at 42.66% in pNIB/PTX-3. (C) Zeta (ξ) potential of the pNIB/PTX complex showed high surface charges of pNIB/PTX micelles, even when the drug is loaded. (D) Photographical data before and after exposure for 24 h at 37 \u003cstrong\u003e°\u003c/strong\u003eC exhibited decreasing transmission pursuant to agglomerate formation of temperature-responsive micelles in the aqueous solution. No precipitate formation was observed in all samples. The graphs on the right side represent the colloidal stability test, which was compared to transmission from 25 \u003cstrong\u003e°\u003c/strong\u003eC to being exposed to 37 \u003cstrong\u003e°\u003c/strong\u003eC for 6, 12 and 24 h. All samples were stable and the most stable condition for the pNIB/PTX-3 complex was taken to be the maximum loading condition.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-1938906/v1/490937905dc75379dcd6a051.png"},{"id":26190830,"identity":"cd1adb58-12a0-489d-91ad-a54a5fa9332a","added_by":"auto","created_at":"2022-09-07 17:19:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":179666,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTemperature-responsive multi-step drug delivery of pNIB/PTX depending on the loading amount of PTX.\u003c/strong\u003e (A) Temperature-dependent transmittance changes according to the loading amounts of PTX. This phenomenon implied a multi-step DDS, in which the primary loaded drug was released by temperature-induced fast delivery with a 23% reduction in transmission at 37 \u003cstrong\u003e°\u003c/strong\u003eC, after which 65% of the remaining drug was slowly delivered by diffusion. (pNIB/PTX-3 shown representatively) (B) The mid-point LCST of the phase transition was found to increase from 36.35–40.17 °C. (C) The high linearity curve of contraction temperature verse amount of loaded PTX, showing adjusting LCST by loaded amount of PTX. (D) PTX loading amount-dependent size changes of pNIB/PTX complex micelles in wet condition (15.63 ppm in PBS) at 25 \u003cstrong\u003e°\u003c/strong\u003eC and 37 \u003cstrong\u003e°\u003c/strong\u003eC, exhibiting properties of multi-step drug delivery via a cascade process.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-1938906/v1/fbc8522cdd2d0f2fedf49252.png"},{"id":26190395,"identity":"8002d6fa-18ad-4063-bde4-52589f989ffb","added_by":"auto","created_at":"2022-09-07 17:09:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":297832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological changes and release profiles based on the temperature responsive characteristics of pNIB/PTX-3.\u003c/strong\u003e (A) Schematic illustration of the multi-step controllable PTX release from pNIB/PTX-3. i) Fast delivery pursuant to partial contraction and slow delivery according to diffusion at body temperature, ii) contraction ratio augmentation depending on the amount of loaded PTX in micelle, resulting in the release of the PTX drug and iii) adjusting the temperature above LCST, followed by allowing rapid drug release that occurs in a short duration or time of need. (B) FE-SEM images of the morphological changes at 25 \u003cstrong\u003e°\u003c/strong\u003eC, 37 \u003cstrong\u003e°\u003c/strong\u003eC, 40 \u003cstrong\u003e°\u003c/strong\u003eC. (C) The histogram showing the size distribution of pNIB and pNIB/PTX at each temperature, exhibiting stepwise contraction (87.76 nm, 63.24 nm, 56.13 nm) owing to increasing LCST (pNIB/PTX-3 shown). Daily and cumulative release profiles of pNIB/PTX at: (D) 37 \u003cstrong\u003e°\u003c/strong\u003eC, (E) 40 \u003cstrong\u003e°\u003c/strong\u003eC. PTX release amounts (μg per mg of pNIB carrier) (left y-axis) and PTX release efficiency (right y-axis) values are presented.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-1938906/v1/31d38078bbbf27f11118a902.png"},{"id":26190680,"identity":"7dddcfe5-7287-436b-9022-0f4e33fd5649","added_by":"auto","created_at":"2022-09-07 17:14:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":135834,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell viability analyses of the human ovarian cancer cells. \u003c/strong\u003eThe cell viabilities of (A) HeyA8 and (B) SKOV3ip1 cells with concentration-dependent experiments of PTX alone, pNIB, and pNIB/PTX-3 groups. Data are shown as the mean ± S.D. (n = 4–8) and fitted with non-linear regression curve fitting (one phase decay).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-1938906/v1/1fc5d41462d14f08f38bb7d4.png"},{"id":26190678,"identity":"00e95584-328d-42a3-a445-b2ecbdb40489","added_by":"auto","created_at":"2022-09-07 17:14:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":113842,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vivo\u003c/em\u003e effects of the pNIB/PTX-3 complex in an orthotopic ovarian cancer mouse model. \u003c/strong\u003e(A) Schematic representation of the \u003cem\u003ein vivo\u003c/em\u003e experimental timeline design. (B) Data of measured tumor weights were analyzed by a box and whisker plot of each experimental group in the mouse models of both HeyA8 and SKOV3ip1. In two groups, 1X pNIB/PTX-3 and 4X pNIB/PTX-3, the tumor weights in the mouse models of both HeyA8 and SKOV3ip1 were significantly decreased. Data is shown as mean ± SD, minimum and maximum values (n = 10). One-way ANOVA followed by Bonferroni post hoc test was used for statistical significance (***p\u0026lt;0.001, ****p\u0026lt;0.0001).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-1938906/v1/fe04880751a89d343d68c11d.png"},{"id":26190399,"identity":"be1e0960-b570-4d0c-bc18-8b216d77bfa4","added_by":"auto","created_at":"2022-09-07 17:09:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":450981,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of tumor detection and apoptotic cell death of pNIB/PTX-3 effects on an orthotopic ovarian cancer mouse model.\u003c/strong\u003e (A and C) Positive staining of Ki67 as a detecting cancer marker was significantly decreased in the pNIB/PTX-3-treated group. (B and D) Apoptotic activity in harvested tumor tissues (SKOV3ip1) was significantly increased in the pNIB/PTX-3-treated group as determined by the terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP nick-end labeling (TUNEL) assay (x100). Data is shown as mean ± SD (n = 11~22). Quantification one-way ANOVA was performed by Tukey's multiple comparisons test and statistical significance is represented with asterisks (**p\u0026lt;0.01, ****p\u0026lt;0.0001).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-1938906/v1/edd94265ad89b1998a2ae308.png"},{"id":26190398,"identity":"83c9cf5c-7e3f-4ff2-aba8-4c6463e69c2a","added_by":"auto","created_at":"2022-09-07 17:09:57","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":88591,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of the pNIB/PTX-3 complex on the mouse survival rates using Kaplan-Meier analysis in SKOV3ip1 model. \u003c/strong\u003eKaplan-Meier analysis of survival data showed that 1X pNIB/PTX-3, 4X pNIB/PTX-3 and PTX alone groups exhibited significantly increased survival of SKOV3ip1 mice model compared with the control group. Statistical significance is indicated by asterisks (*p\u0026lt;0.1).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-1938906/v1/9cc41fd563375c2ec794cb76.png"},{"id":28437264,"identity":"d760e68c-7330-449a-86d0-3f0e8d5be7fd","added_by":"auto","created_at":"2022-10-31 06:24:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2786631,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1938906/v1/89b2a934-fc0a-47d7-a6da-78cc3b5d23a6.pdf"},{"id":26190390,"identity":"a40d39bd-9d6d-4cfb-93a1-245bf3ae6d2e","added_by":"auto","created_at":"2022-09-07 17:09:57","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":303436,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-1938906/v1/b5ccdaf27ac1e0790981419d.png"},{"id":26190396,"identity":"bfc85b40-e43f-4e33-9ef9-4f11f643b650","added_by":"auto","created_at":"2022-09-07 17:09:57","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1471089,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialusshin12220808.docx","url":"https://assets-eu.researchsquare.com/files/rs-1938906/v1/0ee60f4a645dc9fce2e7ade7.docx"}],"financialInterests":"","formattedTitle":"Advanced Drug Delivery of Paclitaxel with Thermoresponsive Water-soluble Micelles for Ovarian Cancer","fulltext":[{"header":"Statement Of Significance","content":"Herein, we developed highly water-soluble and multi-temperature-responsive paclitaxel (PTX) delivery systems for the treatment of ovarian cancer. This was accomplished by complexing the hydrophobic PTX with poly(NIPAAm-co-BVIm) polymeric micelles. In vitro and in vivo analysis showed that our PTX carriers resulted in a six-fold greater therapeutic effect than the only PTX in human ovarian cancer cell and the single injected mice showed similar survival to the mice that were injected weekly with PTX alone with significantly decreased tumor weights. Our multi-step drug delivery system could be novel strategy for the treatment of patients with ovarian cancer."},{"header":"Introduction","content":"\u003cp\u003eEpithelial ovarian cancer\u0026nbsp;is\u0026nbsp;the most\u0026nbsp;lethal disease among various gynecologic\u0026nbsp;malignancies as it is typically diagnosed in the advanced stages. Almost 70~80 % of patients with ovarian cancer are diagnosed with advanced stage\u0026nbsp;including III or IV. These\u0026nbsp;patients diagnosed with metastatic ovarian cancer (stage\u0026nbsp;III) have only a 39% 5-year-survival rate (statistic in the United States from 1975 to 2017)\u0026nbsp;[1].\u0026nbsp;Although most patients with advanced-stage\u0026nbsp;ovarian cancer\u0026nbsp;exhibit poor prognosis,\u0026nbsp;more than 70% of patients\u0026nbsp;exhibit a\u0026nbsp;favorable initial response to\u0026nbsp;standard treatment methods, including maximal debulking\u0026nbsp;surgery and\u0026nbsp;adjuvant\u0026nbsp;chemotherapy\u0026nbsp;with paclitaxel (PTX) and carboplatin\u0026nbsp;[2-4].\u003c/p\u003e\n\u003cp\u003eTaxanes, such as PTX and docetaxel, stabilize polymerized microtubules, resulting in\u0026nbsp;the inhibition of cell division during mitosis\u0026nbsp;[5-9].\u0026nbsp;PTX\u0026nbsp;and carboplatin administered every\u0026nbsp;three weeks are considered the standard first-line chemotherapy for advanced epithelial ovarian cancer.\u0026nbsp;The concept of dose density is based on the hypothesis that a shorter interval between doses of cytotoxic therapy more effectively reduces tumor burden than dose escalation.\u0026nbsp;The duration of exposure is an important determinant of the cytotoxic activity of\u0026nbsp;PTX. A dose-dense schedule leads to frequent exposure of tumor cells\u0026nbsp;[10, 11].\u0026nbsp;Dose-dense\u0026nbsp;(i.e., weekly) delivery of\u0026nbsp;PTX\u0026nbsp;may exploit anticancer mechanisms, such as anti-angiogenesis and the induction of apoptosis\u0026nbsp;[12-14]. Therefore, dose-dense chemotherapy with PTX is currently one of the standard adjuvant therapy options for ovarian cancer\u0026nbsp;[15, 16].\u003c/p\u003e\n\u003cp\u003eFrom the dose-density concept, we hypothesized that continuous release of PTX could increase the duration of exposure to tumor cells and may be an effective ovarian cancer treatment for patients. In addition, even when administered tri-weekly or weekly, PTX has several\u0026nbsp;significant\u0026nbsp;side effects, including peripheral neuropathy and bone marrow suppression, and represents a major limitation in clinical practice\u0026nbsp;[17, 18].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot only do dose density and side effects of PTX represent a certain difficulty in chemotherapy, but also cause problems related to its poor water solubility during the administration.\u0026nbsp;Recently, alternative PTX nanoformulations have been developed to minimize or overcome these limitation\u0026nbsp;[19-24]. Continuous release of PTX through a specific drug delivery system (DDS) that includes a water-soluble complex and a multi-step release process via stimuli-responsive characteristics might be able to decrease PTX-related complications in patients.\u003c/p\u003e\n\u003cp\u003eIn this study, highly water-soluble and temperature-induced PTX delivery systems were developed and examined for their effectiveness in ovarian cancer treatment in a multi-step drug release process. First, we synthesized a highly water-soluble polymeric micelle of poly(NIPAAm-co-BVIm), named pNIB, and then formed a complex with PTX, which is poorly water-soluble. These complexes, pNIB/PTX, could possess highly water-soluble and temperature-responsive characteristics in treatment of ovarian cancer.\u0026nbsp;\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eN\u003c/em\u003e-Isopropylacrylamide (NIPAAm) was purchased from Acros Organics (Geel, Belgium), and purified by recrystallization from hexane (HPLC grade) prior to use. \u003cem\u003eN-\u003c/em\u003evinylimidazole (NVIm), 1-bromobutane, ammonium persulfate (APS), and \u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e\u0026rsquo;,\u003cem\u003eN\u003c/em\u003e\u0026rsquo;-tetramethylethylenediamine (TEMED) were purchased from Sigma\u0026ndash;Aldrich (St. Louis, MI, USA). PTX was purchased from Samyang Co. (Daejeon, Republic of Korea) as a model drug. The Quick Start Bradford Protein assay kit was supplied by Bio-Rad (Hercules, CA, USA). Phosphate-buffered saline (PBS) solution was purchased from Biosesang (Seongnam, Republic of Korea) and used for drug loading and release tests. All other reagents and solvents, purchased from Sigma-Aldrich (St. Louis, MI, USA), were of analytical grade and used without further purification. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Physicochemical and morphologic characterizations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe organic samples were characterized by Fourier-transform infrared spectroscopy (FT-IR) spectroscopy (Spectrum BXII, Perkin-Elmer, Waltham, USA), Zetasizer Nano ZS90 (Malvern, Malvern, UK), and Turbiscan LAB (Formulaction, Toulouse, France). FT-IR spectra were\u0026nbsp;recorded from 4000\u0026ndash;500 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e in 16 scans at 4 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e resolution using potassium bromide (KBr) (Fig. S1). The dispersion stabilities of pNIB/PTX samples in phosphate-buffered saline (PBS) were tested by Turbiscan LAB using a pulsed near-infrared light source (880 nm). The sample solutions (0.125 mg/mL) of 50-mm height were scanned lengthwise every 3 h for 9 h at 37. The light transmitted (or backscattered) by the sample solution at 135 \u0026deg;C\u003csup\u003e\u0026nbsp;\u003c/sup\u003ewas measured.\u003c/p\u003e\n\u003cp\u003eThe 3D morphologies of pNIB micelles with and without PTX were examined by field-emission scanning electron microscopy (FE-SEM, Hitachi S-4300, Hitachi, Tokyo, Japan) after drying the sample solutions on a coverslip at 25 \u0026deg;C and 37 \u0026deg;C. Samples were sputter-coated with approximately 10 nm of gold at a voltage of 15 kV before analysis. The micelle morphologies of the pNIB/PTX micelles with and without PTX in the dry state were characterized by FE-SEM. Each sample solution (0.1 mg) was placed onto a cover slip and then dried at each temperature.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Preparation of pNIB\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;micelles\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eand PTX complexes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 1-butyl-3-vinyl imidazolium bromide- (BVIm) and pNIPAAm-based ionic copolymer poly(NIPAAm-co-BVIm) or pNIB were synthesized as described previously (see the \u003csup\u003e1\u003c/sup\u003eH-NMR spectrum of the pNIB copolymer in Fig. S1) [25].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHydrophilic NIPAAm and BVIm monomers were dissolved in deionized water (DW), APS solution (10% (w/v)) and TEMED were added to the solution as the initiator and activator, respectively.\u0026nbsp;After completion of the reaction, all possible impurities\u0026nbsp;were carefully removed by performing the following steps: i) extraction of organic impurities from the aqueous solution with methylene chloride, ii) removal of inorganic impurities by dialysis (membrane tubing, molecular weight cutoff 12,000\u0026ndash;14,000 Da, Spectrum Laboratories, Savannah, GA, USA) against DW, and iii) repetitive precipitation in water through changes in temperature between 4 and 60 \u0026deg;C. After freeze-drying, the purified copolymer product,\u0026nbsp;pNIB,\u0026nbsp;which looks like white cotton wool, was obtained at\u0026nbsp;a yield of\u0026nbsp;~96.5 wt%. The number- and weight-average molar masses (\u003cem\u003eMn\u003c/em\u003e and \u003cem\u003eMw,\u003c/em\u003e respectively) of the copolymer appeared to be approximately 1353 and 2001 g/mol, respectively, and showed a molar mass distribution (\u003cem\u003eMw\u003c/em\u003e/\u003cem\u003eMn\u003c/em\u003e) of 1.47.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWater-soluble pNIB/PTX complexes were prepared by dissolving 0, 100, 125, 150, 175, and 200 \u0026mu;g PTX in 3 mL of ethyl alcohol containing pNIB (1.0 mg) in an ultrasonic bath at 25 \u0026deg;C for several minutes. Powders of the pNIB/PTX complexes, named pNIB/PTX-1, -2, -3, and -4 in this study, were obtained after filtration and vacuum evaporation of ethyl alcohol at room temperature (Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e \u003cstrong\u003eSummary data for formation of the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003epNIB/PTX\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;complex micelles.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.5%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSample name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.5%\"\u003e\n \u003cp\u003e\u003cstrong\u003epNIB used\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003e\u0026mu;g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.5%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePTX feeded\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003e\u0026mu;g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.5%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePTX loaded\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003e\u0026mu;g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.5%\"\u003e\n \u003cp\u003e\u003cstrong\u003epNIB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.5%\"\u003e\n \u003cp\u003e1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.5%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.5%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.5%\"\u003e\n \u003cp\u003e\u003cstrong\u003epNIB/PTX-1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.5%\"\u003e\n \u003cp\u003e1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.5%\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.5%\"\u003e\n \u003cp\u003e53.38\u0026nbsp;\u0026plusmn;\u0026nbsp;3.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.5%\"\u003e\n \u003cp\u003e\u003cstrong\u003epNIB/PTX-2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.5%\"\u003e\n \u003cp\u003e1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.5%\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.5%\"\u003e\n \u003cp\u003e64.78\u0026nbsp;\u0026plusmn;\u0026nbsp;2.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.5%\"\u003e\n \u003cp\u003e\u003cstrong\u003epNIB/PTX-3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.5%\"\u003e\n \u003cp\u003e1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.5%\"\u003e\n \u003cp\u003e175\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.5%\"\u003e\n \u003cp\u003e74.19\u0026nbsp;\u0026plusmn;\u0026nbsp;2.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.5%\"\u003e\n \u003cp\u003e\u003cstrong\u003epNIB/PTX-4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.5%\"\u003e\n \u003cp\u003e1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.5%\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.5%\"\u003e\n \u003cp\u003e80.16\u0026nbsp;\u0026plusmn;\u0026nbsp;5.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e2.4\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDetermination of loading and releasing amounts of PTX\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe maximum amount of PTX that could be loaded within pNIB copolymer micelles\u0026nbsp;at\u0026nbsp;37\u0026deg;C\u0026nbsp;was determined via solubility (or solution stability) tests in PBS by observation and using TurbiScane LAB. Specifically, 3.0 mg of pNIB/PTX\u0026nbsp;(pNIB/PTX-1, -2, -3, and -4)\u0026nbsp;were separately dissolved in 48 mL PBS solution at room temperature.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e PTX loading and release tests were performed using HPLC calibration curves (Fig. S1). Samples were filtered through a polytetrafluoroethylene (PTFE) membrane filter (JGWP01300, Ominipore\u0026trade;, Millipore, Burlington, MA, USA) with 0.20 \u0026mu;m pores and equipped with a syringe. For the release test, 10 mg of pNIB/PTX-3 sample was dispersed in 10 mL PBS and incubated in a 37 \u0026deg;C water bath with shaking. The PBS solution was filtered daily for 40 d. After filtration, the filtrates were evaporated and re-dissolved in 1 mL acetonitrile, and the resulting solutions (20 \u0026mu;L) were injected into HPLC system. The amount of PTX from the carrier was measured by high-performance liquid chromatography (HPLC, Shimadzu LC20A Series, Kyoto, Japan) at a flow rate of 1 mL/min using an acetonitrile/water solution (50/50 (v/v)) as the mobile phase. (Waters Spherisorb ODS2 column, particle size 5 mm, 4.6 mm 150 mm) PTX was detected at a wavelength of 227 nm. The PTX loading capacity (LC) of pNIB/PTX-3 was calculated using the following equation:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere M\u003csub\u003et\u003c/sub\u003e and M\u003csub\u003e0\u003c/sub\u003e are the cumulative amounts of PTX released at time t and the total amount of PTX entrapped, respectively. The experiment was performed in triplicate.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Measurement of the size distribution and surface charge changes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mean sizes and zeta (\u0026xi;) potential values of pNIB and pNIB/PTX micelles under wet conditions were measured at 25 and 37 \u0026deg;C using Zetasizer Nano ZS90. Sample solutions (3.12 \u0026mu;g/mL) were prepared using a mixture of acetic acid (0.2 M) and sodium acetate (0.1 M). The size distribution curves were obtained using Gaussian curves. These were nearly symmetrical around the vertical line, which passed through the maximum in all cases (Fig. S2). The maximum value is the average size, and the standard deviation is the distance between the two ends at the base. The zeta (\u0026xi;) potential values were recorded in a pH range of 4\u0026ndash;10 or at pH 7 in a mixture of acetic acid (0.2 M) and sodium acetate (0.1 M) using the Zetasizer Nano ZS90 (Fig. S2). The desired pH was adjusted with HCl or NaOH solution, and the pH values were measured using a pH meter (Orion 3 star, Thermo Scientific, Waltham, MA, USA). All measurements were conducted at least three times for each experimental group, and the average values are represented by the standard deviation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDetermination of the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003elower critical solution temperature\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(LCST) values\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe temperature-responsive behaviors (LCST values) of pNIB/PTX micelles with or without PTX were determined using a UV-vis spectrophotometer (Evolution 300, Thermo Scientific, Waltham, MA, USA), fitted with a temperature control system. Aqueous solutions (0.1% wt) of the copolymer were prepared in PBS at pH 7 in ~1 mL of the prepared solution was added to a polystyrene UV cell located in the cell holder. The percentage transmittance of light at 500 nm was scanned for each sample during heating at a rate of 1 \u0026deg;C per 10 min between 25 and 45 \u0026deg;C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 \u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ecell viability assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e cell viability of PTX-free polymer carrier (pNIB), optimized PTX-loaded pNIB (pNIB/PTX-3), and PTX only was evaluated using a Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan) according to the manufacturer\u0026rsquo;s instructions. Human epithelial ovarian cancer (EOC) cell lines, HeyA8 and SKOV3ip1, were a gift from Dr. Anil K. Sood (Department of Cancer Biology, University of Texas M.D. Anderson Cancer Center, TX, USA), and were maintained in complete media (RPMI 1640) supplemented with 10% fetal bovine serum (FBS) and 0.1% gentamicin sulfate (Gemini Bioproducts, Calabasas, CA, USA) in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere at 37 \u0026deg;C. Prior to the cell viability assay, cell suspensions were seeded at a density of approximately 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well in 96-well microplates and pre-incubated for 12 h. The UV-sterilized sample solution was prepared by three-fold serial dilution with complete culture medium. Each well was replaced with each respective concentration set (n = 4\u0026ndash;8) to a final concentration of 1,500 mg/mL to approximately 0.009 mg/mL. PTX alone was diluted from 450 to approximately 0.003 \u0026mu;g/mL. After the plates were incubated at 37 \u0026deg;C for an additional 48 h, particles and floating dead cells were discarded. The plates were then incubated with 100 \u0026mu;L of 10-fold diluted CCK-8 solution for 4 h. The optical density (OD) was measured at 450 nm using a microplate reader (Molecular Devices, USA). Cell viability rates were calculated using the following equation:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere OD\u003csub\u003econtrol\u003c/sub\u003e is obtained in the absence of nanoparticles or drugs. All experiments were repeated four to eight times, and the data are presented as mean \u0026plusmn; standard deviation (SD).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Development of orthotopic mouse models for ovarian cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u0026nbsp;\u003c/em\u003eexperiments were performed to confirm the effects of pNIB/PTX-3 on tumor growth in an orthotopic mouse model using cell lines. Female BALB/c nude mice were purchased from Orient Bio (Seongnam, Republic of Korea). The study was performed in accordance with relevant guidelines and regulations and was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Samsung Biomedical Research Institute (SBRI, Seoul, Republic of Korea). SBRI is an Association for Assessment and Accreditation of Laboratory Animal Care International (protocol no. H-A9-003)-accredited facility and abides by the Institute of Laboratory Animal Resources (ILAR) guidelines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo generate tumors, HeyA8 (2.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells) or SKOV3ip1 (1.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells) in 0.2 mL Hank\u0026apos;s balanced salt solution (HBSS, Gibco, Waltham, MA, USA) were injected into the peritoneal cavity of 8- to 12-week-old BALB/c nude mice. Seven days after the cell injection, 1X pNIB, 4X pNIB, 1X pNIBm/PTX-3 (PTX 120 \u0026mu;g/mouse), 4 X pNIB/PTX-3 (PTX 480 \u0026mu;g/mouse), or PBS only (as a control) was injected into the peritoneal cavity. Mice were monitored daily for tumor development and postoperative complications. Mice were sacrificed on days 35\u0026ndash;40 or when the mice seemed moribund. The body and tumor weights of each mouse were recorded. To assess survival, the animals were observed weekly until death. Tumors were fixed in formalin and embedded in paraffin or snap frozen with liquid nitrogen in OCT compound (Sakura Finetek Japan, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003eApoptotic cell death was assessed according to the manufacturer\u0026rsquo;s instructions using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay kit (Promega, Fitchburg, WI, USA). To quantify cell death, the number of TUNEL positive cells was counted in five random fields at 100x magnification, and the percentage of positive cells was calculated [26].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Statistical analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were generally performed using SPSS ver. 21 (SPSS Inc., Chicago, IL, USA). For \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e analysis, the graphs, calculations, fitting curves and statistical analyses were performed using GraphPad Prism software version 9.0 (GraphPad Software Inc., San Diego, CA, USA). Survival data was plotted via Kaplan-Meier curves and analyzed with the log-rank test. The statistical significance of differences was determined using one-way analysis of variance (ANOVA) for more than two pairs. The level of significance was represented at a \u003cem\u003eP\u003c/em\u003e value of \u0026lt; 0.05 in ANOVA. All experiments were repeated at least three times and data are presented as means \u0026plusmn; standard deviation (S.D.).\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cb\u003e3.1\u0026nbsp;Concept of our study\u0026nbsp;\u003c/b\u003e\n\u003cp\u003eHighly water-soluble and temperature-responsive multi-step drug delivery system for the treatment of ovarian cancer was accomplished by entrapping the hydrophobic PTX molecules within the pNIB polymeric micelles through self-assembly (Fig. 1A). After injection of this pNIB/PTX complex into the ovarian cancer-generated mouse model (Fig. 1B), the complex contracts through drug-regulated lower critical solution temperature (LCST), which allows for the following processes: i) a short-term (a few days) temperature-induced fast release and fast diffusion through the large nanopores of swelled micelles (Fig. 1C, step 2) ii) and then a slow continuous release by diffusion over the long term (about a month) (Fig. 1C, step 3). It is also characterized by a multi-step drug delivery system, which allows the LCST to be downgraded as the drug is released, resulting in phased drug release according to contraction (Fig. 1C, step 4). The physicochemical and biological characteristics of the pNIB/PTX complex, including its high solubility in water, temperature-responsive multi-step delivery system, and advanced therapeutic effects \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e could make it favorable for the stepwise delivery of highly hydrophobic drugs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003cstrong\u003e.2 Formation and water-solubility of the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003epNIB/PTX complexes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe anticancer drug, PTX, which is known to be\u0026nbsp;especially\u0026nbsp;effective for ovarian cancer treatment, has limited delivery capacity\u0026nbsp;through\u0026nbsp;hydrophilic body fluids as it is hydrophobic in nature and exhibits extremely restricted solubility in aqueous media\u0026nbsp;[27, 28].\u0026nbsp;To maximize the\u0026nbsp;delivery efficiency of PTX\u0026nbsp;through\u0026nbsp;aqueous body fluids,\u0026nbsp;water-soluble enhancements and\u0026nbsp;controlled release are necessary\u0026nbsp;[29-35]. To this end,\u0026nbsp;highly water-soluble\u0026nbsp;and temperature-responsive drug-releasable\u0026nbsp;pNIB/PTX -1, -2, -3, and -4\u0026nbsp;complexes\u0026nbsp;were prepared\u0026nbsp;for use in this study as a\u0026nbsp;PTX\u0026nbsp;delivery system for ovarian cancer therapy\u0026nbsp;(summarized in Table 1).\u0026nbsp;This was accomplished by entrapping hydrophobic PTX simultaneously when\u0026nbsp;ionic\u0026nbsp;and\u0026nbsp;highly water-soluble\u0026nbsp;copolymer micelles of pNIB were formed by self-assembly\u0026nbsp;(Fig. 2A).\u0026nbsp;When the amount of PTX loading on each sample was measured, it was confirmed that the amount of PTX loaded during the formation of pNIB polymeric micelles increased proportionally with the amount of feed. The maximum loading amount of PTX was 80.75 \u0026micro;g per 1 mg pNIB polymer. However, the efficiency compared to the feeding amount can be observed to decrease from 53\u0026ndash;40% as the feeding amount increases (Fig. 2B).\u003c/p\u003e\n\u003cp\u003eThe Zeta (\u0026xi;) potential\u0026nbsp;values\u0026nbsp;of pNIB at pH 4\u0026ndash;10 appeared to be in the range +4.8 to +1.8 mV and about\u0026nbsp;+4.2\u0026nbsp;mV at pH 7, indicating that the cationic character of the pNIB copolymer\u0026nbsp;was maintained in various conditions\u0026nbsp;and that it had high solubility in aqueous solution\u0026nbsp;over the full pH range (Fig. S2).\u0026nbsp;Nevertheless,\u0026nbsp;the\u0026nbsp;surface charge of\u0026nbsp;the\u0026nbsp;pNIB/PTX complex changed in accordance with PTX loading, as shown in Fig. 2C.\u0026nbsp;These results clearly indicate that the presence of water-insoluble PTX in all\u0026nbsp;pNIB/PTX\u0026nbsp;complexes decreased the ionic characteristics.\u0026nbsp;Although much lower than the control\u0026nbsp;pNIB\u0026nbsp;due to\u0026nbsp;the hydrophobic characteristic of PTX,\u0026nbsp;this comparison showed similar\u0026nbsp;zeta (\u0026xi;) potential\u0026nbsp;values of approximately 2.7 mV within a margin of error of\u0026nbsp;\u0026plusmn;\u0026nbsp;0.1 mV, indicating that\u0026nbsp;the\u0026nbsp;pNIB/PTX micelle complex was water-soluble after PTX loading.\u003c/p\u003e\n\u003cp\u003eFurthermore, we examinated the dispersion and solubility by recording transmittance at 25 \u0026deg;C and 37 \u0026deg;C for 24 h to determine the stability of each pNIB/PTX solution (Fig. 2D and Fig. S3). During the entire scanning time, no noticeable changes in the light backscattered by pNIB/PTX-1 ~ -3 solutions over time were detected at either 25 or 37 \u003cstrong\u003e\u0026deg;\u003c/strong\u003eC. As the amount of PTX loaded at the two constant temperatures increased, the transmitted light decreased slightly at 25 \u003cstrong\u003e\u0026deg;\u003c/strong\u003eC from ~85% in pNIB/PTX-1 to 70% in pNIB/PTX-4, while at 37 \u003cstrong\u003e\u0026deg;\u003c/strong\u003eC, it decreased significantly from approximately 77% to 45%. This can be explained by the increased hydrophobicity of the solute and the temperature of the solution, which could result in agglomerate formation of colloid (micelle) particles and an increase in the amount of light backscattered. Despite agglomerate formation in the solutions, no precipitate formation was observed in pNIB/PTX-1 ~ -4 in the photographs (Fig. 2D and Fig. S3). These results demonstrate the highly stable and uniform dispersion of pNIB/PTX-1, -2, -3, and -4 complex micelles (or micelle agglomerates) in aqueous solution. Additionally, all samples showed that transmittance increased over time at 37 \u003cstrong\u003e\u0026deg;\u003c/strong\u003eC, indicating that the drug was released and the solubility of pNIB/PTX was improved (Fig. 2D and Fig. S3). Overall, these results should be noted that all samples represent high solubility and dispersion stability, especially for pNIB/PTX-2 and pNIB/PTX-3, which have markedly stable results over time.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003cstrong\u003e.3 Temperature-responsive characteristics of the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003epNIB/PTX complexes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm the temperature-responsive characteristics at 25\u0026nbsp;\u003cstrong\u003e\u0026deg;\u003c/strong\u003eC and 45\u0026nbsp;\u003cstrong\u003e\u0026deg;\u003c/strong\u003eC and at pH 7.0, the\u0026nbsp;LCST values of\u0026nbsp;pNIB/PTX complex\u0026nbsp;were determined and compared with the LCST value of\u0026nbsp;pNIB (Fig. 3A).\u0026nbsp;This can be explained by the increased turbidity of the\u0026nbsp;pNIB\u0026nbsp;solution due to polymer micelle shrinkage caused by the polymer phase transition in the aqueous solution. In contrast, when the contraction degree of pNIB/PTX micelle complex samples was compared by temperature, it was clearly confirmed that the phase transition temperature (LCST) increased above body temperature as the amount of loaded PTX in the micelle increased (Fig. 3A and 3B). Hydrophobic PTX molecules would be primarily attached to\u0026nbsp;the\u0026nbsp;hydrophobic parts\u0026nbsp;of\u0026nbsp;the\u0026nbsp;pNIB\u0026nbsp;micelles such as a (CH\u003csub\u003e2\u003c/sub\u003e-CH\u003csub\u003e2\u003c/sub\u003e)-backbone and isopropyl groups\u0026nbsp; of\u0026nbsp; the\u0026nbsp; pNIB\u0026nbsp; molecules.\u0026nbsp; This interaction between PTX and pNIB caused steric hindrance inside the micelles, resulting in an increased LCST (Fig. S4).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Accordingly, it was confirmed that a linear curve and an R-square value observed at the phase transition temperature according to the amount of loaded PTX were determined by the standard curve in Fig. 3B. Theoretically, the LCST values increased linearly at a rate of 1.16\u003csup\u003e\u0026nbsp;o\u003c/sup\u003eC per 10 \u0026micro;g of PTX out of 1 mg pNIB against the amount of loaded PTX. This means that the temperature-responsive characteristics could be easily changed by adjusting the loading concentration.\u003c/p\u003e\n\u003cp\u003eMoreover, the transmittance of the PTX-free\u0026nbsp;pNIB\u0026nbsp;micelles suddenly decreased by approximately 48% at 37\u0026nbsp;\u003cstrong\u003e\u0026deg;\u003c/strong\u003eC in the LCST range. On the other hand, the transmittance decrease at 37\u0026nbsp;\u003cstrong\u003e\u0026deg;\u003c/strong\u003eC was only approximately 23% for pNIB/PTX-3. This phenomenon also strongly implies a\u0026nbsp;two-step paclitaxel delivery\u0026nbsp;of pNIB/PTX-3: a temperature-induced fast delivery of PTX (approximately 23% of the loaded drug) at 37\u003cstrong\u003e\u0026deg;\u003c/strong\u003eC, followed by slow delivery of the remaining 65% PTX through diffusion at the same temperature\u0026nbsp;(Fig. 1 and Fig. 3A).\u003c/p\u003e\n\u003cp\u003eThese data are also consistent with the results shown in Fig. 3D. As an effective way to confirm whether pNIB micelles contain PTX, the average micelle sizes of pNIB/PTX-1, -2, -3, and -4 complexes in aqueous solution were measured using a Zetasizer at 25 \u003cstrong\u003e\u0026deg;\u003c/strong\u003eC and 37 \u003cstrong\u003e\u0026deg;\u003c/strong\u003eC and compared to the values of PTX-free pNIB complex with a temperature increase of 25 \u003cstrong\u003e\u0026deg;\u003c/strong\u003eC to 37 \u003cstrong\u003e\u0026deg;\u003c/strong\u003eC, the series of pNIB/PTX samples showed temperature-induced decreases in the average size of the complex micelles (129, 48, 27.9, 4.2 nm for pNIB/PTX-1, -2, -3, -4, respectively). The PTX-free pNIB micelles exhibited the largest drop of approximately 391 nm. These results indicate that the sharp drop in the diameter of the pNIB/PTX series at 25 \u003cstrong\u003e\u0026deg;\u003c/strong\u003eC is caused by the effective integration of hydrophilic pNIB micelles with hydrophobic PTX molecules. When the PTX concentration increased, the hydrophilicity and equilibrium swelling degree of the pNIB/PTX complexes decreased, resulting in a decrease in the diameter. In particular, the temperature-induced decrease in diameter of approximately 27.9 nm observed from pNIB/PTX-3 micelles might be appropriate for multi-step paclitaxel delivery. This result demonstrates fast delivery at 37 \u003cstrong\u003e\u0026deg;\u003c/strong\u003eC, followed by slow delivery by diffusion at the same temperature. In addition, the opposite is possible if the LCST increases as the drug is loaded. As shown in Fig. 3D, the smaller the amount of drugs loaded, the smaller the size of the drug carrier at the same temperature, 37 \u003cstrong\u003e\u0026deg;\u003c/strong\u003eC. This means that as the drug is released, the amount of drug loaded in the micelle decreases, thereby reducing the LCST gradually through a cascade process, further accelerating drug release (Fig. S4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003cstrong\u003e.4\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eIn vitro release profile of PTX from the pNIB/PTX-3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this respect, pNIB/PTX-3 was selected as a\u0026nbsp;representative model because it has the maximum loading condition considering the loading capacity and efficiency and highly stable uniform dispersion. Most importantly, its LCST is suitable for application in ovarian cancer via multi-step anti-cancer drug delivery.\u003c/p\u003e\n\u003cp\u003eThe stepwise temperature-responsive release process of the pNIB/PTX-3 complex was observed by FE-SEM for morphological changes depending on the temperature, with or without PTX (Fig. 4A).\u0026nbsp;Upon increasing the amount of\u0026nbsp;loaded PTX\u0026nbsp;within the pNIB/PTX complex\u0026nbsp;at a constant temperature, the following morphological changes were observed: i) the shape changed spherical particles (pNIB) into a raspberry shape (pNIB/PTX-3) according to the drug loaded, ii) the average size of pNIB/PTX-3 became smaller than that of PTX-free pNIB micelles.\u0026nbsp;In addition, the histogram showing the size distribution of each sample by temperature indicated the temperature range in which the complexes contracted (Fig. 4B). For instance, pNIB contracted to 43.85\u0026nbsp;nm at 37\u0026nbsp;\u003csup\u003eo\u003c/sup\u003eC, which had an average size similar to one at 40\u0026nbsp;\u003csup\u003eo\u003c/sup\u003eC\u0026nbsp;(41.65\u0026nbsp;nm). In contrast,\u0026nbsp;pNIB/PTX-3\u0026nbsp;contractions of the hydrogel sequentially occurred (87.76 nm, 63.24, and 56.13 nm) at 25\u0026nbsp;\u003csup\u003eo\u003c/sup\u003eC, 37\u0026nbsp;\u003csup\u003eo\u003c/sup\u003eC and 40\u0026nbsp;\u003csup\u003eo\u003c/sup\u003eC.\u0026nbsp;As PTX was loaded, the LCST increased, resulting in a sequential size reduction at increasing temperatures (Fig. 4A, Fig. S5 and Table S1.). This analysis to be a consistent result with the above figure, and it is worth noting that pNIB/PTX-3 has a consistent contraction ratio with Fig. 3A, especially given that it has a contraction ratio of 27.94% from 25\u0026nbsp;\u003csup\u003eo\u003c/sup\u003eC\u0026nbsp;to 37\u0026nbsp;\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 4C and 4D, the temperature-dependent cumulative release amount of PTX was investigated at 37 \u0026deg;C and 40 \u0026deg;C. All samples showed sustained release profiles\u0026nbsp;for up to\u0026nbsp;40 days. The initial rapid release\u0026nbsp;(0.59 %/day, 4.41\u0026nbsp;\u0026plusmn; 0.04\u0026nbsp;\u0026mu;g/day\u0026nbsp;from 1 mg pNIB/PTX-3)\u0026nbsp;for the first five days\u0026nbsp;may have been caused\u0026nbsp;by fast diffusion of hydrophobic PTX molecules through the relatively large nanopores of the swelled micelles and relatively weak compressive desorption of PTX molecules through temperature-responsive micelle contraction at 37\u0026deg;C,\u0026nbsp;whereas the\u0026nbsp;relatively slow\u0026nbsp;release\u0026nbsp;(0.11 %/day,\u0026nbsp;0.81 \u0026plusmn; 0.06 \u0026mu;g/day\u0026nbsp;from 1 mg pNIB/PTX-3)\u0026nbsp;for the remaining days\u0026nbsp;may have been caused\u0026nbsp;by the decreased diffusion speed of drug molecules because of reduced loading amount and narrowed nanopores of the micelles, and by relatively fast compressive desorption of PTX because of lowed LCST values of the micelles at the same temperature. At 40\u0026nbsp;\u0026deg;C, the drug release was markedly more rapid for 5 d (0.63 %/day, 4.68\u0026nbsp;\u0026mu;g/day\u0026nbsp;from 1 mg pNIB/PTX-3). And also,\u0026nbsp;the cumulative\u0026nbsp;maximum release amounts\u0026nbsp;were more increased (750.1\u0026nbsp;\u0026mu;g) than 37\u0026nbsp;℃ (512.2\u0026nbsp;\u0026mu;g)\u0026nbsp;for the entire period.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTaken together, when the temperature increased near the body temperature (37 \u0026deg;C), the resulting temperature-induced delivery occurred sequentially: fast delivery by size contraction and then slow delivery by diffusion. This process is a multi-step drug delivery process that is required for sustained release. In addition, when the temperature was raised above the LCST (40 \u0026deg;C), the pNIB/PTX complex shrank more, resulting in an instant greater release of the drug. Therefore, the micelle complex can control the amount of drug release, as desired in the right place.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003cstrong\u003e.5 \u003cem\u003eIn vitro\u003c/em\u003e dose-dependent therapeutic effects of the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003epNIB/PTX-3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the potential therapeutic effects of pNIB/PTX-3 \u003cem\u003ein vitro\u003c/em\u003e, two human ovarian cancer cell lines, HeyA8 and SKOV3ip1, were treated with different concentrations of\u0026nbsp;PTX,\u0026nbsp;pNIB,\u0026nbsp;and pNIB/PTX-3\u0026nbsp;at 37\u0026nbsp;\u003cstrong\u003e\u0026deg;\u003c/strong\u003eC\u0026nbsp;for 2 d. Cell viability was assessed using CCK-8 assay (Fig.\u0026nbsp;5).\u003c/p\u003e\n\u003cp\u003eExposure to\u0026nbsp;only\u0026nbsp;the\u0026nbsp;PTX-carrier\u0026nbsp;(pNIB)\u0026nbsp;had IC\u003csub\u003e50\u003c/sub\u003e values of 11.89 \u0026mu;g/mL with approximately 75% cell viability in HeyA8 cells and 33.09 \u0026mu;g/mL with about 65% cell viability in SKOV3ip1 cells. The IC\u003csub\u003e50\u003c/sub\u003e values depending on treatment with pNIB/PTX-3 and PTX alone were 1.516 and 233 \u0026mu;g/mL, respectively, with about 70% and 40% cell viability in HeyA8 cells, in which the calculated PTX concentrations were 0.311 \u0026mu;M and 273 \u0026mu;M, respectively (Figure\u0026nbsp;5A). In SKOV3ip1 cells, the IC\u003csub\u003e50\u003c/sub\u003e values of pNIB/PTX-3 and PTX alone were 3.421\u0026nbsp;mg/mL (~40%) and 136.2\u0026nbsp;mg/mL (~30%), respectively, and the calculated PTX concentrations were 0.701 \u0026mu;M and 159 \u0026mu;M, respectively (Fig.\u0026nbsp;5B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere was a greater reduction in cell viability in SKOV3ip1 cells (~36%) relative to HeyA8 (~55%) with pNIB/PTX-3. HeyA8 cells had an approximate 55% cell viability with 5.556 \u0026mu;g/mL of pNIB/PTX-3 and in the PTX treatment when PTX was loaded at ~1.1 \u0026mu;M and a concentration of PTX was 6.5 \u0026mu;M. This demonstrates that pNIB/PTX-3 in both cell types had six times more therapeutic effect than PTX treatment alone. We observed dose-dependent inhibitory effects of pNIB/PTX-3 on ovarian cancer cell viability. These results suggest that the pNIB/PTX-3 had good therapeutic effects on ovarian cancer cells and could be an ideal candidate material for PTX delivery in human ovarian cancer patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003cstrong\u003e.6 \u003cem\u003eIn vivo\u0026nbsp;\u003c/em\u003eassay of the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003epNIB/PTX-3\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003efor ovarian cancer therapy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the potential clinical relevance of the \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eresults, we performed \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003eexperiments using ovarian cancer orthotopic mouse models. HeyA8 and SKOV3ip1 ovarian cancer cells were implanted into the peritoneal cavity of female nude mice and 7 days after cell injection, and therapy was initiated according to the following five treatment regimens: control (PBS saline), 1X pNIB, 4X pNIB, 1X pNIB/PTX-3, and 4X pNIB/PTX-3 (Fig. 6A). In the carrier alone group, which included 1X pNIB and 4X pNIB, tumor weight did not differ from the control. Thus, carrier pNIBIm had no influence on the mouse tumor weight. However, mice treated with PTX loaded with pNIBIm, which included 1X pNIB/PTX-3 and 4X pNIB/PTX-3, had significantly decreased tumor weight compared to the control and carrier alone groups using either of the cell types (Fig. 6B and 6C).\u003c/p\u003e\n\u003cp\u003eWe also found that there was no significant difference between 1X pNIB/PTX-3 and 4X pNIB/PTX-3. Thus, 1X pNIB/PTX-3 is a sufficient dose for the treatment of tumor-bearing mice. Daily monitoring of animals throughout the course of therapy showed acceptable tolerability with no adverse side effects, such as changes in body weight, mobility, posture, or feeding habits. Because PTX has anti-proliferative and pro-apoptotic activity in cancer cells, we examined tumor cell proliferation and apoptosis using Ki67 expression and a TUNEL assay, respectively, in harvested tumor tissues. Positive staining of Ki67 was significantly decreased in the pNIB/PTX-3-treated group compared to that in the controls (Fig. 7A and 7C, P \u0026lt; 0.01). A TUNEL assay also showed significantly increased apoptosis in the treated groups (Fig. 7B and 7D, P \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003eFinally, we evaluated the survival of mice after injection in the SKOV3ip1 mouse model. At 7 d post-injection, therapy was started according to the following six treatment regimens: control, 1X pNIB, 4X pNIB, 1X pNIB/PTX-3, 4X pNIB/PTX-3, and PTX alone. We administered a single i.p. injection to the four pNIB-treated groups, but PTX 120 mg/mouse, i.p. once a week in the PTX alone group. Kaplan-Meier analysis showed that the 1X pNIB/PTX-3, 4X pNIB/PTX-3, and PTX alone groups had significantly increased survival rates compared to the control group (Fig. 8). However, among the three PTX-containing treatment groups, 1X pNIB/PTX-3 and 4X pNIB/PTX-3 showed no significant differences from the PTX alone group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe pNIB/PTX complex showed six times higher therapeutic effect with a single i.p. injection than weekly PTX treatment alone in \u003cem\u003ein vitro\u003c/em\u003e experiments using two human ovarian cancer cell lines, HeyA8 and SKOV3ip. Moreover, we found that the singly injected complex significantly decreased the tumor growth in orthotopic mouse models using either of the cell lines. Survival was significantly prolonged in the mice compared to the control and showed survival rates similar to those obtained by weekly injections of PTX alone. These results suggest that a single injection of pNIB/PTX-3 has a survival benefit similar to that of weekly PTX injections.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eHerein, we successfully developed highly water-soluble and multi-step drug-releasing\u0026nbsp;pNIB/PTX complexes\u0026nbsp;by\u0026nbsp;entrapping water-insoluble PTX (53.38–80.16 μg) within highly water-soluble and temperature-responsive pNIB micelles (1.0 mg).\u0026nbsp;While the\u0026nbsp;high colloidal stability and uniform dispersion of\u0026nbsp;pNIB/PTX-1, -2, and\u0026nbsp;-3\u0026nbsp;complexes in aqueous solution were confirmed, the\u0026nbsp;pNIB/PTX-3\u0026nbsp;sample showed\u0026nbsp;optimal\u0026nbsp;solution stability and maximum PTX-loading capacity at 37 °C.\u0026nbsp;Highlight\u0026nbsp;temperature-induced multi-step PTX delivery\u0026nbsp;confirmed that the LCST increased above body temperature in accordance with the increasing amount of loaded PTX in micelles. In particular,\u0026nbsp;pNIB/PTX-3 micelle complex are efficient for the\u0026nbsp;temperature-induced fast delivery of the loaded PTX (approximately 23%) at 37 °C, followed by the slow delivery of the remaining PTX (approximately 65%) through diffusion at 37 °C. Furthermore,\u0026nbsp;the average\u0026nbsp;micelle\u0026nbsp;sizes\u0026nbsp;of\u0026nbsp;complex samples, with and without PTX,\u0026nbsp;in aqueous solution\u0026nbsp;from 25–37 °C\u0026nbsp;exhibited a strong reduction\u0026nbsp;in diameter. These results strongly indicate that pNIB/PTX-3 may be controlled by\u0026nbsp;a cascade process\u0026nbsp;involving the gradual reduction of LCST. The SEM images\u0026nbsp;of pNIB/PTX-3 at 25 °C, 37 °C, and 40 °C\u0026nbsp;also appeared\u0026nbsp;very different in terms of particle shape (rough raspberry-shaped and dense particle-shaped, respectively) and diameter (Contraction 30% at 37 °C and 40% at 40 °C). In addition, unlike pNIB, which already exhibited a contraction at 37 °C, progressive contraction was characterized by morphological transition at 25 °C, 37 °C, and 40 °C. During\u0026nbsp;the\u0026nbsp;40 d\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e PTX\u0026nbsp;release assay\u0026nbsp;at\u0026nbsp;37 °C,\u0026nbsp;an initial rapid release\u0026nbsp;(0.59 %/day, 4.41\u0026nbsp;± 0.04 μg/day from 1 mg pNIB/PTX-3)\u0026nbsp;was observed for the first five days, which was followed by a\u0026nbsp;slow\u0026nbsp;release\u0026nbsp;(1.1 %/day, 0.81 ± 0.06\u0026nbsp;μg/day from 1 mg pNIB/PTX-3)\u0026nbsp;for the remaining days.\u0026nbsp;Therefore, the drug was released more rapidly (75.01 μg per 1 mg pNIB/PTX-3) at temperatures higher than the LCST (40\u0026nbsp;°C)\u0026nbsp;when compared to the drug release at 37 °C (51.22 μg per 1 mg pNIB/PTX-3), resulting in a sudden drug release. Taken together, the highly water-soluble pNIB/PTX-3 complexes demonstrate multifunctional properties, including a high loading amount of PTX, outstanding water solubility, and multi-step drug release in accordance with temperature, and may be used as promising ovarian cancer-target materials.\u003c/p\u003e\n\u003cp\u003eDuring the \u003cem\u003ein vitro\u003c/em\u003e experiments\u0026nbsp;of the pNIB/PTX-3\u0026nbsp;complex\u0026nbsp;on the\u0026nbsp;two ovarian cancer cell lines,\u0026nbsp;HeyA8 and SKOV3ip1, the PTX carrier complex, pNIB/PTX-3,\u0026nbsp;showed\u0026nbsp;a six times\u0026nbsp;stronger\u0026nbsp;therapeutic effect than\u0026nbsp;PTX\u0026nbsp;treatment alone.\u0026nbsp;These inhibitory effects of pNIB/PTX-3 suggest that the\u0026nbsp;PTX carrier\u0026nbsp;had good therapeutic effects on ovarian cancer cells and could be an ideal candidate biomaterial for\u0026nbsp;PTX\u0026nbsp;delivery.\u0026nbsp;In the orthotopic ovarian cancer mouse models, this complex significantly decreased tumor weight compared to controls in both the HeyA8 and SKOV3ip1 models. Moreover, this\u0026nbsp;singly injected\u0026nbsp;complex significantly prolonged mouse survival compared to controls and showed similar survival to mice\u0026nbsp;treated with weekly injections of\u0026nbsp;PTX alone.\u003c/p\u003e\n\u003cp\u003eThese results show that\u0026nbsp;the\u0026nbsp;good\u0026nbsp;water-solubility and temperature-induced\u0026nbsp;multi-step\u0026nbsp;PTX release of the pNIB/PTX complex\u0026nbsp;are highly\u0026nbsp;useful\u0026nbsp;for\u0026nbsp;short-term and long-term delivery of highly hydrophobic anticancer drugs,\u0026nbsp;including PTX, to\u0026nbsp;ovarian cancer\u0026nbsp;cells.\u003c/p\u003e\n\u003cp\u003eDespite the great advantages associated with the use of PTX in cancer therapy, its administration has certain limitations due to its poor solubility and low permeability. In this study, we successfully developed advanced delivery systems based on stimuli-responsive PTX release to address this limitation. Our multi-step drug delivery system showed ovarian cancer-targeting effect, making it a good therapeutic candidate for the treatment of patients with ovarian cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u0026nbsp;\u003c/strong\u003e: All animal studies performed in this study were in accordance with relevant guidelines and regulations and was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Samsung Biomedical Research Institute (SBRI, Seoul, Republic of Korea). Consent : not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e : Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e: The datasets used and/or analyzed during the current study are available from the corresponding author, upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u0026nbsp;\u003c/strong\u003e: The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e: This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) [2020R1F1A1072210]. This work was also supported by the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health \u0026amp; Welfare, Republic of Korea, the Ministry of Food and Drug Safety) [KMDF_PR_20200901_0153-2021] and the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) [2020R1C1C1007482].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor’s contributions\u0026nbsp;\u003c/strong\u003e: Prof. Ueon Sang Shin, Prof. Jeong-Won Lee : conceived and supervised. Ji-Hye Kang, Ji-Young Hwang, Young Jae Cho : wrote and performed most of the experimental studies. Sang-Yu Park, Jung-Joo Choi, E-Sun Paik : research co-ordination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e: This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) [2020R1F1A1072210]. This work was also supported by the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health \u0026amp; Welfare, Republic of Korea, the Ministry of Food and Drug Safety) [KMDF_PR_20200901_0153-2021] and the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) [2020R1C1C1007482].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHowlader N NA, Krapcho M, Miller D, Brest A, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA SEER Cancer Statistics Review, 1975-2017. J Natl Cancer Inst Bethesda, MD. 2020.\u003c/li\u003e\n\u003cli\u003eArmstrong DK, Bundy B, Wenzel L, Huang HQ, Baergen R, Lele S, et al. Intraperitoneal cisplatin and paclitaxel in ovarian cancer. N Engl J Med. 2006,354:34-43.\u003c/li\u003e\n\u003cli\u003eCortez AJ, Tudrej P, Kujawa KA, Lisowska KM. 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ACS Appl Mater Interfaces. 2018,10:25174-85.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"drug delivery system (DDS), paclitaxel (PTX), temperature-responsive polymeric micelle, ovarian cancer","lastPublishedDoi":"10.21203/rs.3.rs-1938906/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1938906/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"As an anti-cancer drug, paclitaxel (PTX) is known to be effective for treating patients with ovarian cancer. Not only do dose density and side effects of PTX represent a certain difficulty in chemotherapy, but also cause problems related to its poor water solubility during the administration. From the dose-density concept, we hypothesized that continuous release of PTX could increase the duration of exposure to tumor cells and may be an effective ovarian cancer treatment for patients. Herein, we developed highly water-soluble and multi-temperature-responsive PTX delivery systems to treat ovarian cancer. This was accomplished by entrapping the hydrophobic PTX in poly(NIPAAm-co-BVIm) micelles engineered to deliver drugs (pNIB/PTX). Specifically, the pNIB/PTX-3 complex containing 74 μg PTX loaded at 25 °C showed a rapid release of PTX initially (5.9 %/day) during the first five days at 37 °C. The release rates decreased (1.1 %/day) over the following month, indicating a multi-step release. In orthotopic ovarian cancer mouse models, the pNIB/PTX complex resulted in a six-fold greater therapeutic effect with a single intraperitoneal injection than weekly PTX treatment alone in vitro using two human ovarian cancer cell lines, HeyA8 and SKOV3ip. Moreover, the tumor weights in HeyA8 and SKOV3ip1 models were remarkably decreased with the pNIB/PTX complex compared to the controls. The good water solubility and temperature-dependent release of PTX carriers are highly effective for short-term and long-term delivery. This multi-step drug delivery may be used as a potential candidate for the treatment of patients with ovarian cancer.","manuscriptTitle":"Advanced Drug Delivery of Paclitaxel with Thermoresponsive Water-soluble Micelles for Ovarian Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-09-07 17:09:55","doi":"10.21203/rs.3.rs-1938906/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cc77040f-1e79-446a-a897-2c90e2cd949a","owner":[],"postedDate":"September 7th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-10-31T06:24:36+00:00","versionOfRecord":[],"versionCreatedAt":"2022-09-07 17:09:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1938906","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1938906","identity":"rs-1938906","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}